Cholinesterase is a generic term for those enzymes that hydrolyze choline ester to choline and an organic acid.
In the body exist true cholinesterase and pseudocholinesterase. True Ch--E exists in red blood-corpuscle, in the nervous-system, as well as in muscles and has an effect to decompose specifically acetyl choline or acetyl-.beta.-methylcholine. On the other hand, pseudo Ch--E exists in serum, in liver and pancreas, and it decomposes specifically benzoyl-choline and mainly the cholinester of fatty acids with a longer chain of carbon atoms, such as for example butyrylcholine. Of these two kinds of Ch--E, the quantitative determination of the activity of pseudo Ch--E especially in serum is of highest importance, for the examination of liver disease, because the activity of pseudo Ch--E decreases greatly at times of disease of the liver, especially in cases of chronic damages of liver parenchyma and liver cirrhosis. In addition, such a determination is of importance for the treatment of poisonings with phosphorous containing compounds used in agriculture and furthermore for examination at therapeutic treatment by means of an anti-cholinesterase preparation.
For determination of Ch--E activity, gas-analysis, .DELTA. pH-method, colorimetric analysis, UV-procedure, fluorescent procedure and electrode-method are known. In cases of clinical routine examinations, .DELTA. pH-method and colorimetric analysis are most conventional. In case of .DELTA. pH-method in which acetic acid formed by Ch--E effect is determined by means of a pH-indicator, a complicated correction of the strongly deviated calibration-curve is necessary and the accuracy is uncertain.
Among colorimetric analysis, there are the DTNB-procedure (Garrv. D. J., Clin. Chem.11.(2):91 (1965)) and the enzyme procedure (Kunihide Gomi: Rinshobyori Special nunber 29: 145(1977)).
In case of DTNB procedure, acetyl thiocholine, butyryl-thiocholine are used as substrate and the thus formed thiocholine is mixed with 5.5'-dithio-bis-2-nitrobenzoic acid (DTNB) and the density of yellow colour in comparison with glutathione in reduced form as standard of a SH-compound is measured spectrometrically. The exactness of the determination is therefore interfered by bilirubin and a reducing substance and furthermore the stability of the reagents during storage is not satisfactry.
In case of enzyme-procedure, benzoyl choline and ortho-toluoyl-choline are used as substrate and the choline formed by enzyme reaction is changed to betaine with choline oxidase. By the action of H.sub.2 O.sub.2 thus formed, an oxidizing condensation of 4-aminoantipirin with phenol in presence of peroxidase occurs with colouring that has to be measured spectrometically. The determination is therefore influenced by additional choline that is formed through decomposition of phospholipid in serum, because choline shall be converted with choline oxidase. Besides, such determination is interfered by reducting substances such as bilirubin and ascorbic acid. Furthermore, this process is not usuful for determination by means of an auto-analyser.
To overcome the disadvantages of known processes, a method to assay Ch--E activity in serum was suggested by the applicant of this invention according to Japanese patent application No. 093896/1980. In this method p-hydroxybenzoic-acid that is formed from p-hydroxybenzoyl-choline as a substrate through enzymatic effect of Ch--E and has been oxidatingly condensed with 4-aminoantipyrin in presence of an oxidizing agent to obtaining a coloured compound and the degree of colouring is being measured.